Human immunodeficiency virus (HIV) sequence diversity is a major hurdle for the reliable assessment of anti-viral T cell immunity in vitro and for the design of an effective and broadly applicable HIV vaccine. See Brander C, et al., Curr. Opin. Immunol. 2006; 18(4):430-437.
Despite the extensive HIV sequence diversity on a full genome level, the variation in individual positions of viral proteins is often restricted to a limited number of amino acid substitutions that recur or “toggle” throughout the different HIV clades. See Gaschen B, et al., Science 2002; 296:2354-2360. Toggling residues often include amino acid pairs such as arginine (R) and lysine (K) or leucine (L) and valine (V), which are biochemically similar and are, therefore, likely to be tolerated by the virus without serious impacts on its replication efficiency. Remarkably, most of Gag p24—representative of a highly conserved protein-toggled peptide mixtures only require fewer than 20 variants (median 4), even at the highest level of sequence coverage (i.e. only amino acids present in <5% of database sequence are excluded from toggle positions), while the number of variants is higher in other Gag subunits at this coverage. See Yusim K, et al., J. Virol. 2002; 76(17): 8757-8768. In an IFN-γ Elispot assay, it had been shown that these “toggle” peptides detect HIV specific CD4+ and CD8+ T cell responses of significantly higher breath and magnitude than matched consensus peptides. See Frahm N, et al., J. Immunol. 2007; 179:6638-6650.
Most of the studies of immunogen candidates for HIV vaccination are based in the analysis of IFN-γ production by Elispot. Until now, no direct correlation between magnitude or secretion of IFN-γ and viral load has been found. During the last years, it has been shown that the quality of the T cell responses against HIV antigenic determinants may be the key determinant in eliciting an effective response against the virus. However, the analysis of poly-functional responses (e.g. different cytokine secretion and perforate exocytose) of CD8+ T lymphocytes in HIV infected patients have shown a negative correlation between the polifunctionality capability of the T lymphocytes and viral load. In addition, flow cytometry and Elispot-based analyses may not detect responses associated to cytokines that are not measured regularly or that do not show expected effector function profile(s). Consequently, there is a need in the art for more sensitive and comprehensive methods for evaluating T cell response against HIV.